Modification of the rat airway explant transcriptome by cigarette smoke.

Although a number of animal model studies have addressed changes in gene expression in the parenchyma and their relationship to emphysema, much less is known about the pathogenesis of cigarette smoke-induced small airway remodeling. In this study the authors exposed rat tracheal explants, a model of the airway wall, to whole smoke for 15 min, and then cultured the explants in air. The airway transcriptome was evaluated using RAE 230_2 gene chips. By 2 h after starting smoke exposure, expression levels of 502 genes were differentially expressed by more than 1.5 times (p < .01 or less) and by 24 h 1870 genes were significantly changed up or down. These included genes involved in antioxidant protection, epithelial defense and remodeling, inflammatory mediators and transcription factors, and a number of unexpected genes, including the matrix metalloproteinase (MMP)-12 inducer, tachykinin-1 (substance P). Pretreatment of the explants with 1 x 10(-7) M dexamethasone reduced the number of significantly changed genes by approximately 47% at 2 h and 68% at 24 h and in almost all instances reduced the magnitude of the smoke-induced changes. The authors conclude that even a very brief exposure to cigarette smoke can lead to rapid changes in the expression of a large number of genes in rat tracheal explants, and that these effects are directly mediated by smoke, without a need for exogenous inflammatory cells. Steroids, contrary to the usual belief, are able to ameliorate many of these changes, at least in this very acute model.

A model to identify novel targets involved in oxidative stress-induced apoptosis in human lung epithelial cells by RNA interference.

Chronic obstructive pulmonary disease (COPD) is an increasing health problem primarily associated with cigarette smoking, and one of the leading causes of morbidity and mortality worldwide. Despite recent advances in understanding the pathogenesis of the disease, overall patient outcome remains poor with limited therapeutic intervention. Chronic inflammation, an imbalance between proteolytic and anti-proteolytic activities (leading to lung parenchyma destruction) and excessive oxidative stress contribute to COPD pathophysiology. Oxidative stress-triggered apoptosis of alveolar structural cells, including epithelial cells, may be an important event in the development of COPD. In this study, we developed a cell-based oxidative stress-induced apoptosis assay and performed a high-throughput screen (HTS) using a human druggable genome siRNA library. Our results have identified potential novel pathways (e.g. unfolded protein response, proteosomal activity) and targets (e.g. MAP3K14, HMGB2) that regulate the response of lung epithelial cells to oxidative stress. This assay has proven to be a useful tool for the identification of potential new therapeutic targets for lung disease.

Macrolides as immunomodulatory medications for the therapy of chronic lung diseases.

Macrolide antibiotics have potent immunomodulatory activity. The spectrum of action of these antibiotics extends to regulation of leukocyte function and production of inflammatory mediators, control of mucus hypersecretion, resolution of inflammation, and modulation of host defense mechanisms. Macrolides are now being used or investigated to treat chronic lung inflammatory diseases, including diffuse panbronchiolitis (DPB), cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD) and asthma. Intense research is ongoing to further elucidate the targets and mechanism/s of action of macrolides in eukaryotic cells. In this paper, we review recent findings on novel effects of macrolides on epithelial barrier function and resolution of inflammation, which may shed light on the mechanisms underlying the beneficial effects of macrolides in the clinic.

Clarithromycin has an immunomodulatory effect on ERK-mediated inflammation induced by Pseudomonas aeruginosa flagellin.

OBJECTIVES: Pseudomonas aeruginosa exoproducts are potent triggers of immune responses in eukaryotic cells. Clarithromycin initially decreases, then increases and finally produces a sustained suppression of interleukin (IL)-8 secretion from normal human bronchial epithelial (NHBE) cells through inhibition and activation of extracellular signal-regulated kinase (ERK). This polyphasic immune response is referred to as immunomodulation. METHODS: We studied the effects of P. aeruginosa flagellin and alginate on IL-8 secretion from NHBE cells and how this was affected by clarithromycin or dexamethasone. We also assessed the upstream kinase cell signalling intermediates that appear to be responsible for flagellin-induced IL-8 secretion. ELISA was used to measure IL-8 in culture supernatants, and western blots were used to measure kinase and phosphokinase levels. RESULTS: Flagellin dose-dependently increased IL-8 secretion in NHBE cells at 24 h, whereas alginate had no effect on IL-8. Clarithromycin significantly decreased flagellin-induced IL-8 over the first 9 h but not at 24 h. A 60 min exposure to clarithromycin decreased flagellin-induced ERK phosphorylation, but at 24 h, clarithromycin increased phospho-ERK1/2 beyond the effect of flagellin alone. Pre-treatment with PD98059 (MEK inhibitor) decreased flagellin-induced IL-8 secretion by 47.7% (P < 0.0001) compared with control flagellin exposure and decreased basal IL-8 in the absence of flagellin by 27.9% compared with untreated control cells (P < 0.0001). Dexamethasone and PD98059 together had an additive suppressive effect on flagellin-induced IL-8 secretion. CONCLUSIONS: P. aeruginosa flagellin, but not alginate, stimulates IL-8 secretion in NHBE cells in part through ERK1/2. This effect is modulated by clarithromycin, whereas suppression of IL-8 secretion by dexamethasone probably occurs through different pathways.

Bordetella bronchiseptica flagellin is a proinflammatory determinant for airway epithelial cells.

Motility is an important virulence phenotype for many bacteria, and flagellin, the monomeric component of flagella, is a potent proinflammatory factor. Of the three Bordetella species, Bordetella pertussis and Bordetella parapertussis are nonmotile human pathogens, while Bordetella bronchiseptica expresses flagellin and causes disease in animals and immunocompromised human hosts. The BvgAS two-component signal transduction system regulates phenotypic-phase transition (Bvg+, Bvg-, and Bvg(i)) in bordetellae. The Bvg- phase of B. bronchiseptica is characterized by the expression of flagellin and the repression of adhesins and toxins necessary for the colonization of the respiratory tract. B. bronchiseptica naturally infects a variety of animal hosts and constitutes an excellent model to study Bordetella pathogenesis. Using in vitro coculture models of bacteria and human lung epithelial cells, we studied the effects of B. bronchiseptica flagellin on host defense responses. Our results show that B. bronchiseptica flagellin is a potent proinflammatory factor that induces chemokine, cytokine, and host defense gene expression. Furthermore, we investigated receptor specificity in the response to B. bronchiseptica flagellin. Our results show that B. bronchiseptica flagellin is able to signal effectively through both human and mouse Toll-like receptor 5.

Pseudomonas aeruginosa flagellin and alginate elicit very distinct gene expression patterns in airway epithelial cells: implications for cystic fibrosis disease.

Infection with the opportunistic pathogen Pseudomonas aeruginosa remains a major health concern. Two P. aeruginosa phenotypes relevant in human disease include motility and mucoidy. Motility is characterized by the presence of flagella and is essential in the establishment of acute infections, while mucoidy, defined by the production of the exopolysaccharide alginate, is critical in the development of chronic infections, such as the infections seen in cystic fibrosis patients. Indeed, chronic infection of the lung by mucoid P. aeruginosa is a major cause of morbidity and mortality in cystic fibrosis patients. We have used Calu-3 human airway epithelial cells to investigate global responses to infection with motile and mucoid P. aeruginosa. The response of airway epithelial cells to exposure to P. aeruginosa motile strains is characterized by a specific increase in gene expression in pathways controlling inflammation and host defense. By contrast, the response of airway epithelia to the stimuli presented by mucoid P. aeruginosa is not proinflammatory and, hence, may not be conducive to the effective elimination of the pathogen. The pattern of gene expression directed by flagellin, but not alginate, includes innate host defense genes, proinflammatory cytokines, and chemokines. By contrast, infection with alginate-producing P. aeruginosa results in an overall attenuation of host responses and an antiapoptotic effect.

Neutrophil serine proteinases cleave bacterial flagellin, abrogating its host response-inducing activity.

After bacterial infection, neutrophils dominate the cellular infiltrate. Their main function is assumed to be killing invading pathogens and resolving the inflammation they cause. Activated neutrophils are also known to release a variety of molecules, including the neutrophil serine proteinases, extracellularly. The release of these proteinases during inflammation creates a proteolytic environment where degradation of different molecules modulates the inflammatory response. Flagellin, the structural component of flagella on many bacterial species, is a virulence factor with a strong proinflammatory activity on epithelial cells and other cell types. In this study we show that both human and mouse neutrophil serine proteinases cleave flagellin from Pseudomonas aeruginosa and other bacterial species. More important, cleavage of P. aeruginosa flagellin by the neutrophil serine proteinases neutrophil elastase and cathepsin G resulted in loss of the biological activity of this virulence factor, as evidenced by the lack of innate host defense gene expression in human epithelial cells. The finding that flagellin is susceptible to cleavage by neutrophil serine proteinases suggests a novel role for these enzymes in the inflammatory response to infection. Not only can these enzymes kill bacteria, but they also degrade their virulence factors to halt the inflammatory response they trigger.